Agent derived from tortoise spleen stimulating mammalian hemopoiesis

ABSTRACT

The present invention relates to a proteinaceous extract derived from tortoise spleen and to a tetrapeptide FTGN, which have stimulatory activity on hematopoietic cells. In particular, this tetrapeptide enhances hemopoietic reconstruction, and bone marrow re-population, reduced as a consequence of a high dose of radiation or chemotherapy exposure. The invention further provides pharmaceutical compositions comprising as an effective ingredient the proteinaceous extract or the FTGN tetrapeptide and ex vivo and in vivo methods of treatment employing them.

FIELD OF THE INVENTION

The present invention relates to a proteinaceous agent derived fromtortoise spleen, and to an oligopeptide that stimulates theproliferation of mammalian hemopoietic cells. More specifically, thesecompounds enhance engraftment of bone marrow transplant, hemopoieticreconstruction and bone marrow re-population and therefore can be usedfor curing and/or alleviating the detrimental effect of ionizingradiation and/or cytotoxic chemicals on a tissue or body. The inventionfurther provides methods for using this tetrapeptide and pharmaceuticalcompositions comprising it.

BACKGROUND OF THE INVENTION

All publications mentioned throughout this application are fullyincorporated herein by reference, including all references citedtherein.

Exposure to ionizing radiation has detrimental effects on tissues; andexposure of hemopoietic cells to such radiation, may provoke lifethreatening consequences. Radiation therapy is an important regimen ofmany anticancer treatments, together with chemotherapy, where cytotoxiceffects of both these therapies often affect hemopoiesis. Therefore,protecting agents could substantially improve the efficiency of currentanticancer therapies, in addition to their possible use in cases ofaccidental irradiation.

Radiotherapy is a treatment for cancer and other diseases thatincorporates ionizing radiation to destroy malignancies. Ionizingradiation damages or destroys cells in the area being treated,preventing the malignant cells from continuing to grow and multiply.Most radiotherapy techniques employ high energy X-rays or sometimesGamma rays. In some instances internal radiotherapy (e.g. radioactiveimplant placed inside the body) may be used.

Radiotherapy can damage normal cells as well as cancer cells, and theremay be potential side effects, which would depend on the radiotherapydose, site(s) of treatment, age and other factors. The side effects ofradiation therapy include temporary or permanent loss of hair in thearea being treated, skin irritation, temporary change in skin color inthe treated area, and tiredness.

Since radiation therapy can be and often is used in combination withchemotherapy or surgery, other common side effects as fatigue, pain,nausea, vomiting, decreased blood cell counts, hair loss, and mouthsores may worsen the patient's condition and also increase the patient'sdiscomfort.

One of currently used protections against radiation damages is thetransplantation of bone marrow or administration of peripheral bloodstem cells in early days post irradiation or chemotherapy. However, thehematopoietic stem cells recover to only 5% to 10% of normal levelsafter bone marrow transplantation [Mauch P. et al.: Blood (1989) Vol.74(2):972; Thorsteinsdottir U. et al.: Blood (1999) Vol. 94 (8):2605],and recovery time is too long [Vellenga E. et al.: British J. Haematol.(1987) Vol. 65(2):137], not mentioning the problems of bone marrowstorage [Soderdahl G. et al.: Bone Marrow Transplant (1998) Vol.21(1):79].

The ability to modulate differentiation and proliferation ofhematological precursors is at the basis of the more innovativetherapies such as peripheral blood stem cell transplant, genetransfection and ex vivo expansion of stem cells. In spite of thisimpressive progress, several aspects of stem cell physiology have notbeen fully clarified.

Several factors are suspected of being involved in the physiological orpathological proliferation/differentiation of bone marrow cells. Inaddition to the role of classically defined growth factors, severalbiological agents and cell types could improve or modify both in vivoand ex vivo therapeutic strategies. Human bone marrow-derivedendothelial cells support long term proliferation and differentiation ofmyeloid and megakaryocytic progenitors [Rafii, S., et al., Blood (1995)Vol. 86:353]; accessory cells may support hematological recovery afterbone marrow transplant [Bonnet, D., et al., Bone Marrow Transpl. (1991)Vol. 23:203].

Short peptides have been synthesized to reach hemoregulatory andmultilineage effects, possibly by enhancement of cytokine production bystromal cells [King, A. G., et al., Exp. Hematol. (1992) Vol. 20(4):531;Pelus, L. M., et al., Exp. Hematol. (1994) Vol. 22:239].

For example, the osteogenic growth peptide (OGP) was shown to induce, invivo, a balanced increase in white blood cell (WBC) counts, and overallbone marrow cellularity in mice receiving myeloablative irradiation andsyngeneic or semiallogeneic bone marrow transplants [Gurevitch, O., etal., Blood (1996) Vol. 88(12):4719].

Therefore, substances that can induce increment in colony forming units(CFU) capacity of bone marrow cells and related cells along thedifferent differentiation paths, should find clinical application intreatments intending to restore the hematopoietic cells damaged bychemotherapeutic agents and/or radiation.

Oligopeptides that support hemopoiesis may prove useful in other ways aswell. Some investigators have found that adding stem cells fromperipheral blood to those from bone marrow significantly increases therate of engraftment. However, extracting sufficient numbers of stemcells from peripheral blood is a complicated procedure. Administeringsuch oligopeptides to donors to increase the number of stem cells in theblood will improve the feasibility of transplanting stem cells fromperipheral blood [Golde, D. W., Sci. Am. (1991) Vol. 36 (December)].

The capacity of the hematopoietic stem cells to provide for the lifelongproduction of all blood lineages is accomplished by a balance betweenthe plasticity of the stem cell, that is the production of committedprogenitor cells which generate specific blood lineages, and thereplication of stem cell in the undifferentiated state (self-renewal).The mechanisms regulating hematopoietic stem cells plasticity andself-renewal in vivo have been difficult to define. However, the majorcontributory factors represent a combination of cell intrinsic andenvironmental influences [Morrison, et al., Proc. Natl. Acad. Sci. USA(1995) Vol. 92:10302].

A prerequisite for hemopoiesis and therefore successful BMT is thepresence of functional stromal cells and tissue that form part of thehemopoietic microenvironment, determine the homing of the injected stemcells from the circulation to the bone marrow and support hemopoiesis[Watson, J. D. and McKenna, H. J. Int. J. Cell Cloning (1992) Vol.10:144]. Growth of bone marrow cells is supported by the stromal tissue.The tissue components provide the conditions needed for the survival ofstem cells in long-term in vitro bone marrow cultures. At present thistechnology suffices to keep stem cells alive. Adding an appropriatefactor, e.g. a “hemopoietic” oligopeptide to these cultures may helpexpand the stem cell population ex vivo/in vitro, thus providingincreased numbers of these cells for transplantation.

A combined in vitro/in-vivo approach may provide the basis for aforward-looking strategy for (i) obtaining small stem cell preparationsfrom donors of blood or marrow and (ii) enabling healthy individuals tohave their stem cells stored for any future therapeutic need, thusbypassing the complexity associated with the use of allogeneic BMT.

It would therefore be of therapeutic importance to use small peptidessuch as the oligopeptide described in the present application, thatstimulate post-BMT hemopoietic reconstruction by enhancing in vivo, exvivo and/or in vitro the progenitor hemopoietic cells.

A largely used protective agent is amifostine [Merck Index, 12th Ed.], athiophosphate developed by the US army as a radioprotective agent, andcurrently used to decrease the cytotoxic effects of both radiationtherapy and chemotherapy. Amifostine must be administered shortly beforeirradiation; once reconstituted, its stability at room temperature isquite limited [Gosselin T. K. and Mautner B.: Clin. J. Oncology Nursing(2002) Vol. 6:175]. Most patients are afflicted by some of many sideeffects of amifostine, which include, e.g., hypotension, allergies,nausea and vomiting, the latter two occurring in approximately 53% ofpatients [Gosslin and Mautner, Ibid.].

The development of a non-toxic selective protective agent thatpreferentially protects normal tissues from chemotherapy toxicity,without protecting malignant tissues, is a major challenge in cancerchemotherapy research. The available protective agents are either toxicor lack selective protective activity.

It is therefore an object of this invention to provide a new protectiveagent, conferring protection to a tissue or body exposed to a cytotoxicfactor, such as ionizing irradiation or cytotoxic chemical.

The tortoise is a quite remarkable animal in that it can survive a doseof ionizing radiation greater than other vertebrata, and nearly100-times greater than mammals (Table 1) [Khamidov D. K. et al.: Bloodand Haemopoiesis of Vertebrates with Radiation Injuries, Monograph,Tashkent (1986), “FAN” 175 pp.]. TABLE 1 Lethal doses of ionizingradiation for different animals Animal Lethal dose - LD_(50/30) (Gy)Golden fish 25 Frog 15 Lizard 25 Tortoise 500 Pigeon 15 Mouse 7

EP 0377044 B1 describes a protective effect of a nonapeptide (EAKSQGGSN)on irradiated mice. U.S. Pat. No. 5,866,160 describes a composition ofsoft-shelled turtle and tortoise, for enhancing the leukocyte number inpatients undergoing chemotherapy. Russian Patent RU 2118533C1 describesan extract from tortoise liver for improving hematopoietic function inirradiated mammals, and for increasing their viability. Jacobson[Jacobson L. O. et al.: Proc. Soc. Exptl. Biol. Med. (1950) Vol. 73:455]demonstrated an important role of spleen by screening the said organduring irradiation of mice, and finding that their survival ratesignificantly increased.

It was found that intraperitoneal injection of tortoise plasma increasedthe survival rate of mice after whole body γ-irradiation (8 Gray; Gy)and that the injection of a spleen extract bad a still stronger effect[Turdiev A. et al.: Radiobiology (1985) Vol. 25:655; Turdiev A. et al.:Radiation Biology and Radioecology (1998) Vol. 38:63].

It is therefore another object of this invention to provide a newprotective agent conferring protection to a tissue or body exposed to acytotoxic factor, such as an ionizing irradiation or a cytotoxicchemical, wherein said protective agent is derived from tortoise spleen.

It is further an object of this invention to provide a pharmaceuticalcomposition comprising a factor or oligopeptide derived from tortoisespleen, that can decrease damages caused by an ionizing irradiation to atissue or body, wherein said irradiation is either accidental or a partof radiation therapy.

It is a still further object of this invention to provide apharmaceutical composition comprising a factor or oligopeptide derivedfrom tortoise spleen that can decrease damages caused to a tissue orbody by an exposure to a cytotoxic chemical, wherein said exposure iseither accidental or a part of chemotherapy.

Other objects and advantages of present invention will become apparentas description proceeds.

SUMMARY OF THE INVENTION

The present invention relates to a proteinaceous composition of matterderived from tortoise spleen capable of stimulating the proliferation ofmammalian hemopoietic cells.

In a preferred embodiment, the composition of matter of the inventioncomprises an oligopeptide having molecular weight up to 2000 Daltons,preferably an oligopeptide that comprises from 4 to 20 amino acids, andparticularly an oligopeptide comprising the amino acid sequence FTGN(SEQ ID: No. 1).

The composition of matter of the invention stimulates the proliferationof mammalian hemopoietic cells, enhances hemopoiesis in an irradiatedsubject, increases spleen mass, increases bone marrow count, and/orimproves the probability of survival of said irradiated subject. Moreparticularly, the composition of matter of the invention increases bonemarrow cells (BMC) count in an irradiated subject followingtransplantation of bone marrow cells which have been exposed to saidcomposition of matter before being transplanted into said irradiatedsubject.

The invention further relates to an oligopeptide comprising the aminoacid sequence FTGN (SEQ ID: No. 1), preferably an oligopeptide havingthe amino acid sequence FTGN (SEQ ID: No. 1) or its biologicallyfunctional variants, modifications and derivatives, its physiologicallyacceptable salts, esters and amides of said oligopeptide and derivativesthereof that maintain the ability to stimulate the proliferation ofmammalian hemopoietic cells,

In a further aspect the invention relates to biologically functionalvariants of the oligopeptide of the invention, wherein such variant maycomprise an alteration in the side chain of an amino acid of saidoligopeptide, resulting from either in vivo mutation or from chemicalmodification in vitro; a dimer or multimer of said oligopeptide.

The invention also relates to the oligopeptide modifications whereinsaid modification or derivative comprises a chemical modification ofside chains of the amino acids, or modification of the terminal carboxylor amino groups, alkylation, acylation, amidation, or esterification, inwhich said modification modulates the biological activity of saidoligopeptide, and/or improves its stability in vivo.

In a special embodiment, the invention relates to a protectivecomposition for in vivo treatment, preferably by injection, comprisingan oligopeptide or physiologically acceptable salts, esters, and amidesof said oligopeptide or its variants, modifications, and derivatives,for reducing the detrimental effect of a cytotoxic factor selected fromionizing radiation and cytotoxic chemicals on a tissue or body exposedthereto, wherein the exposure of tissue or body to ionizing radiation orcytotoxic chemical is accidental or a part of radiation therapy orchemotherapy. Said composition being useful for in vitro or ex vivotreatment of hemopoietic cells.

In another aspect, the invention describes a pharmaceutical compositioncomprising an oligopeptide as active ingredient, preferably atetrapeptide having the amino acid sequence FTGN (SEQ ID: No. 1), itsphysiologically acceptable salts, esters, and amides of saidoligopeptide or its functional variants, modifications, and derivativesfurther comprising pharmaceutically acceptable carriers, excipientsand/or diluents. Said pharmaceutical composition may further comprise anadditional active agent selected from growth factors, anti-rejectionagents or tolerance inducing agents, analgesics, antibiotics,anti-inflammatory agents, antineoplastics, cyto-protectants,glucocorticoids, hematopoietics, and immunosuppressants.

In a preferred embodiment, the invention relates to a method forreducing the detrimental effect of a cytotoxic factor selected fromionizing radiation and cytotoxic chemicals on a mammalian subjectexposed thereto, comprising administering to said subject a compositionof matter or an oligopeptide or a physiologically acceptable salt,ester, or amide thereof or a variant, modification, or derivativethereof. This method comprises the steps of: obtaining hemopoietic cellsof said mammal; ex vivo treatment of said hemopoietic cells with thecomposition of matter of the invention, or with an oligopeptideaccording to the invention or with physiologically acceptable salts,esters, and amides thereof or with functional variants, modifications,and derivatives thereof as previously defined; and re-implanting saidcells into said mammal wherein treated cells are donor's hemopoieticcells.

In a different embodiment, the method of the invention may be appliedfor reducing the detrimental effect of a cytotoxic factor selected fromionizing radiation and cytotoxic chemicals on a tissue or body of amammalian subject exposed to said factor, wherein the exposure of saidtissue or subject to ionizing radiation or cytotoxic chemical/factor isaccidental or a part of a radiation/chemotherapy, by directlyadministering to said mammal a composition of matter, or an oligopeptideor physiologically acceptable salts, esters, and amides thereof orfunctional variants, modifications, and derivatives thereof as definedabove.

In a preventive approach, the method of the invention may comprise aninitial step wherein cells are treated with said composition of matteror said peptide or said derivative before the exposure to said cytotoxicfactor.

The invention also relates to the use of the composition of matter, oroligopeptide, or physiologically acceptable salts, esters, and amidesthereof or variants, modifications, and derivatives thereof, allaccording to the invention, in the preparation of a medicament forreducing the detrimental effect of a cytotoxic factor selected fromionizing radiation and cytotoxic chemicals on a tissue or body of amammal exposed thereto.

In a more specific embodiment, the invention relates to the use of atetrapeptide having the amino acid sequence FTGN (SEQ ID: No. 1), apharmaceutically acceptable salt thereof, or a functional derivatethereof obtained by esterification, amidation, alkylation, or acylation,in the preparation of a medicament.

The invention thus encompasses a composition of matter, an oligopeptideor physiologically acceptable salts, esters, and amides thereof orfunctional variants, modifications, and derivatives thereof, for use asa medicament for reducing the detrimental effect of a cytotoxic factorselected from ionizing radiation and cytotoxic chemicals on a tissue orbody of a mammal exposed thereto.

More specifically, the active factor of said medicament is atetrapeptide having the amino acid sequence FTGN (SEQ ID: No. 1), orpharmaceutically acceptable salts or physiologically acceptablefunctional derivatives thereof comprising alkylation, acylation,amidation, or esterification, of said tetrapeptide.

In another embodiment, the invention relates to an oligopeptidecomprising the amino acid sequence FTGN (SEQ ID: No. 1), preferably atetrapeptide having the amino acid sequence FTGN (SEQ ID: No. 1) orphysiologically acceptable salts, esters, and amides thereof, or itsvariants, modifications, and derivatives maintaining the capability tostimulate the proliferation of mammalian hemopoietic cells, for use as acyto-protective agent.

The above and other characteristics and advantages of the invention willbe more readily apparent through the following examples, and withreference to the appended drawings, wherein:

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: HPLC Chromatogram of Tortoise Spleen Extract

Different separated fractions were obtained after HPLC: fractions a-h.

Abbreviations: Fract.: fraction; T: time; Min: minutes.

FIG. 2: Biological Activity of the Different Tortoise Spleen ExtractsHPLC Fractions

Irradiated mice (6 Gy) were injected with 2 μg of the purified fractionsd-h. The hemopoietic activity in the mice was evaluated at the 14^(th)day post-irradiation by assessing the number of colonies originated fromspleen and bone marrow cells. The fraction treatment results arecompared to control mice (irradiated but not treated) or PBS mice(irradiated and injected with PBS buffer).

Abbreviations: Spl. Fract.: spleen fraction; Sp.: spleen; BM: bonemarrow; CFU: colony forming units; Cont.: control.

FIG. 3 a-d: Biological Activity of the Tortoise Spleen Extract HPLCFraction a.

Bone marrow cells were treated ex vivo with fraction a or PBS andtransplanted into irradiated mice.

FIG. 3 a: Bone marrow smear of an irradiated mouse without anytreatment.

FIG. 3 b: Bone marrow smear of an irradiated mouse transplanted with BMCtreated with PBS.

FIG. 3 c: Bone marrow smear of an irradiated mouse transplanted with BMCtreated with the spleen extract fraction a.

FIG. 3 d: Spleen morphology of the mice from FIGS. 3 a-3 c as seen atthe ninth day after irradiation. Left: control mice, Middle: micetransplanted with BMC treated with PBS, Right: mice transplanted withBMC previously treated with fraction a.

FIG. 4: HPLC Chromatogram Sub Fractionation of Fraction a.

Active spleen extract fraction a was further purified to sub-fractionsa1-a6.

Abbreviations: T: time; Min: minutes.

FIG. 5: TLC Chromatogram Comprising Fractions a4 to a6.

Thin layer chromatography of sub-fractions a4, a5 and a6. Fraction a5shows the presence of oligopeptides. Control amino acids Glu, Val, Gly,Met and Tyr were included.

FIG. 6 a-c: FTGN Tetrapeptide Stability.

FIG. 6 a: HPLC chromatogram of FTGN tetrapeptide reconstituted in waterafter storage at −10° C. for one year.

FIG. 6 b: HPLC chromatogram of normal blood serum

FIG. 6 c: HPLC chromatogram of FTGN tetrapeptide reconstituted in serumand incubated at 37° C. during 4 hours.

Abbreviations: T: time; Min: minutes; AU: arbitrary units.

FIG. 7 a-c: FTGN Tetrapeptide Biological Activity Tested In Vitro.

BM cells were incubated in vitro in RPMI medium in the presence (10μg/ml) or absence of FTGN tetrapeptide for 2 hours. Cells were washedand cloned in methylcellulose-containing medium.

FIG. 7 a: BM cells from non irradiated mice incubated in the presence(+) or absence (−) of FTGN tetrapeptide.

FIG. 7 b: BM cells from irradiated mice (4.5 Gy) 24 hours previous tothe incubation in the presence (+) or absence (−) of FTGN tetrapeptide.

FIG. 7 c: BM cells from non irradiated mice, irradiated ex vivo (4.5 Gy)and incubated in the presence (+) or absence (−) of FTGN tetrapeptide.

Abbreviations: CFU: colony forming units.

FIG. 8 a-b: Spleen Morphology of Irradiated Mice Transplanted with BMCPre-Treated with FTGN.

Total body irradiated mice (7.0 Gy) were injected with 6×10⁴ BMCpreviously treated with FTGN tetrapeptide.

FIG. 8 a: Spleens from mice transplanted with FTGN treated BMC, 4, 9 and16 days after transplant.

FIG. 8 b: Spleens from mice transplanted with untreated BMC, 4, 9 and 16days after transplant

Abbreviations: T: time; D: days.

FIG. 9: Spleen Weight of Irradiated Mice Transplanted with BMCPre-Treated with FTGN.

Total body irradiated mice (7.5 Gy) were injected with 6×10⁴ BMCpreviously treated with FTGN tetrapeptide at different concentrations(0.1, 0.5, 1.0 and 10.0 μg/ml). Spleen weight was measured 9 (darkcolumns) and 16 (clear columns) days after irradiation and compared tocontrol mice (normal non-irradiated mice) or PBS mice (mice transplantedwith BMC incubated with PBS).

Abbreviations: Treat.: treatment; BMC: bone marrow cells; W: weight; mg:milligrams; Cont.: control.

FIG. 10: Spleen CFU of Irradiated Mice Transplanted with BMC Pre-Treatedwith FTGN.

Total body irradiated mice (7.5 Gy) were injected with 6×10⁴ BMCpreviously treated with FTGN tetrapeptide at different concentrations(0.1, 0.5, 1.0 and 10.0 μg/ml). Spleen colonies were measured 9 daysafter irradiation and compared to PBS mice (mice transplanted with BMCincubated with PBS).

Abbreviations: Treat.: treatment; BMC: bone marrow cells; CFUs: colonyforming units from spleen.

FIG. 11: BMC Count in Irradiated Mice Transplanted with BMC Pre-Treatedwith FTGN.

Total body irradiated mice (7.5 Gy) were injected with 6×10⁴ BMCpreviously treated with FTGN tetrapeptide at different concentrations(0.1, 0.5, 1.0 and 10.0 μg/ml). Bone marrow cells were counted 9 (lightcolor) and 16 (dark color) days after irradiation and compared to PBSmice (mice transplanted with BMC incubated with PBS).

Abbreviations: Treat.: treatment; BMC: bone marrow cells and BM cellcount (10⁶: millions).

FIG. 12: Bone Marrow CFU of Irradiated Mice Transplanted with BMCPre-Treated with FTGN.

FIG. 12: Bone marrow CFU

Total body irradiated mice (7.5 Gy) were injected with 6×10⁴ BMCpreviously treated with FTGN tetrapeptide at different concentrations(0.1, 0.5, 1.0 and 10.0 μg/ml). BM colonies were measured 9 (lightcolor) and 16 (dark color) days after irradiation and compared tocontrol mice (irradiated but not transplanted mice) or PBS mice (micetransplanted with BMC incubated with PBS).

Abbreviations: Treat.: treatment; BMC: bone marrow cells; CFU BM: colonyforming units from BMC; Cont.: control.

FIG. 13: WBC Count in Irradiated Mice Transplanted with BMC Pre-Treatedwith FTGN.

Total body irradiated mice (7.5 Gy) were injected with 6×10⁴ BMCpreviously treated with FTGN tetrapeptide at different concentrations(0.1, 0.5, 1.0 and 10.0 μg/ml). White blood cells were counted 16 daysafter irradiation and compared to control mice (irradiated but nottransplanted mice) or PBS mice (mice transplanted with BMC incubatedwith PBS).

Abbreviations: Treat.: treatment; BMC: bone marrow cells; WBC: whiteblood cells (10⁶/ml); Cont.: control.

FIG. 14: Spleen CFU of Irradiated Mice Transplanted with BMC Pre-Treatedwith Various FTGN Concentrations.

Total body irradiated mice (7.0 Gy) were injected with 6×10⁴ BMCpreviously treated with FTGN tetrapeptide at different concentrations(0.1, 0.5, 1.0, 10.0 and 50.0 μg/ml). Spleen colonies were measured 9days after irradiation and compared to mice transplanted with BMCincubated only with PBS.

Abbreviations: Treat.: treatment; CFU: colony forming units from spleen.

FIG. 15: BMC Count in Irradiated Mice Transplanted with BMC Pre-Treatedwith Various FTGN Concentrations.

Total body irradiated mice (7.0 Gy) were injected with 6×10⁴ BMCpreviously treated with FTGN tetrapeptide at different concentrations(0.1, 0.5, 1.0, 10.0 and 50.0 μg/ml). Bone marrow cells from femur werecounted 4, 9 and 16 days after irradiation and compared to micetransplanted with BMC incubated only with PBS.

Abbreviations: Treat.: treatment; BMC: BM cell count (10⁶/b:millions/bone).

FIG. 16 a-b: BMC Count in Irradiated Mice Transplanted with BMCIrradiated/Non Irradiated Donor Cells Pre-Treated with FTGN.

Donor BM cells incubated ex vivo in RPMI medium in the presence (10μg/ml) or absence of FTGN tetrapeptide for 2 hours were transplantedinto irradiated recipient mice (7.0 Gy.). Twenty days after transplantBMC/bone were counted.

FIG. 16 a: transplanted BM cells from irradiated (4.5 Gy) donor miceincubated in the presence (+) or absence (−) of FTGN tetrapeptide.

FIG. 16 b: transplanted BM cells from non irradiated control miceincubated in the presence (+) or absence (−) of FTGN tetrapeptide.

Abbreviations: BMC: BM cell count (10⁶/b: millions/bone).

FIG. 17 a-c: BMC Count in Irradiated Mice Treated with FTGN.

Irradiated mice (6.5 Gy) were treated in vivo with the FTGNtetrapeptide. Thirty days after treatment BMC were counted.

FIG. 17 a: Control mice irradiated and injected with PBS.

FIG. 17 b: Irradiated mice I.V. injected once with 50 μg FTGN 2 hoursafter irradiation.

FIG. 17 c: Irradiated mice I.V. injected twice with 50 μg FTGN (eachtime) 2 and 24 hours after irradiation.

Abbreviations: BMC: bone marrow cell count (10⁶/b: millions/bone);Treat.: treatment; I.V.: intravenous.

FIG. 18 a-c: Bone Marrow CFU Count of Irradiated Mice Treated with FTGN.

Irradiated mice (6.5 Gy) were treated in vivo with the FTGNtetrapeptide.

Thirty days after treatment CFU from BM were counted

FIG. 18 a: Control mice irradiated and injected with PBS.

FIG. 18 b: Irradiated mice I.V. injected once with 50 μg FTGN 2 hoursafter irradiation.

FIG. 18 c: Irradiated mice I.V. injected twice with 50 μg FTGN (eachtime) 2 and 24 hours after irradiation.

Abbreviations: CFU BM: colony forming units from BMC; Treat.: treatment;I.V.: intravenous.

FIG. 19: Survival Curve of Irradiated Mice Transplanted with BMCPre-Treated with Purified a5 Spleen Extract Subfraction.

Irradiated mice (8 Gy) were transplanted with BMC previously incubatedwith the spleen extract fraction a5. Mice survival was pursue during 30days and compared to irradiated mice untreated (PBS) or mice that weretransplanted with untreated donor BMC.

Abbreviations: BMC: bone marrow cells; An. N.: animal number, TPR: timepost-radiation; D: days.

FIG. 20: Survival Curve of Irradiated Mice Transplanted with BMCPre-Treated with FTGN.

Lethally irradiated mice (10 Gy.) were transplanted with 60.000 donor BMcells previously incubated for 1 hour in the presence (10 mg/ml) orabsence of FTGN tetrapeptide. Mice survival was pursue during 30 daysand compared to control irradiated mice untreated or mice that weretransplanted with untreated donor BMC.

Abbreviations: TAT: time after treatment, D: days; % Surv.: percentageof mice survival; Cont.: control.

DETAILED DESCRIPTION OF THE INVENTION

The inventor has found that certain HPLC fractions of tortoise spleenextract have a surprisingly strong protective effect on irradiated mice,and further it has been found that said effect was related to aproteinaceous factor in said fractions. Recovery of white cellspopulations in surviving mice has been found to be consistently enhancedby said proteinaceous factor. The results indicated that said tortoisespleen proteinaceous factor was responsible for proliferation ofundifferentiated stem cells that had survived irradiation in mice, andfor their differentiation into lymphoid and myeloid cell lines, whichcould be observed in the bone marrow from about 8^(th) day postirradiation. Said factor was assumed to be responsible for thesurprising radio-resistance in mice, further confirmed by the increasedsurvival rate of the irradiated mice.

This tortoise spleen proteinaceous factor has been linked witholigopeptides of molecular weight below 2000 Daltons. In a preferredpurification protocol, the extract from tortoise spleen is fractionatedby protein and peptide chemistry techniques known in the art. Suchpeptide purification techniques may include: gel filtrationchromatography, ion-exchange chromatography, affinity chromatography,reverse phase chromatography, HPLC with C18 column, electrophoresis, TLCand MS.

The proteinaceous factor or the oligopeptide of this invention areobtained either by extraction from tortoise spleen, or by synthesis,using techniques known in the art of protein chemistry.

The proteinaceous factor and the oligopeptide of this invention areoriginated from natural compounds and therefore, they are considerednon-toxic. This fact may allow the repeated use of the compounds, asneeded according to the situation of the subject in need, withoutconcern regarding secondary effects.

A tetrapeptide isolated from the HPLC fractions of the tortoise spleenextract showed the same biological properties as seen for the wholetortoise extract described above (Examples 1, 3, 4 and 5). Thistetrapeptide amino acid sequence is identified as FTGN (SEQ ID: No. 1)

The term oligopeptide and tetrapeptide used through all the inventiondescription are equally related to the synthetic FTGN (SEQ ID: No. 1)and refer to an isolated peptide.

The tetrapeptide was synthesized and it was found to support hemopoiesisin irradiated mice. Mice that were whole-body irradiated with a dose of7.5 Gy (which is normally considered a lethal dose), were transplantedby intravenous injection with donor's bone marrow cells preincubated exvivo with different concentrations of the synthetic tetrapeptide. Thecomparison between treated and untreated mice showed that tetrapeptideFTGN enhanced spleen growth, substantially enhanced the number of spleencolonies, and increased the number of bone marrow cells (BMC) and whiteblood cells (WBC), as described in Examples 4 and 5.

In adult mice the spleen is a hematopoietic active organ, especiallyfollowing recovery from damage inflicting treatment, such asirradiation. Transplanted cells “home” to the spleen (as well as the BM)and first develop into discrete colonies, which later become confluent.This results in an increase in the spleen size. As the hematopoieticsystem recovers, the spleen returns into its normal size. Therefore,FTGN tetrapeptide treatment may well accelerate hematopoietic recoveryof the spleen. Mice transplanted with FTGN-treated cells demonstrated anearly increment in the spleen size, a faster development of spleencolonies and an earlier normalization of the spleen size.

It is understood that all proteinaceous tortoise spleen factorssupporting hemopoiesis, irrespective of their derivation, are a part ofthis invention. It is further understood that any tortoise-derivedproteinaceous composition is a part of this invention, which comprises apeptide or protein comprising amino acid sequence FTGN, or its variantsmaintaining the above described protective activity or a derivativethereof or a modification maintaining the above described protectiveactivity, i.e. functional derivatives. Said functional derivatives ormodifications may include, but are not limited to, chemicalmodifications, such as substitution of side chains of amino acids, oralternatively modification of the terminal carboxyl or amino groups orinternal hydroxyl, wherein the modification may comprise amidation,esterification, and alkylation. Said modification may, e.g., modulatethe biological activity, or improve stability, such as in vivo stabilityin plasma after injection or chemical stability, or otherwise. Anoligopeptide, comprising in its sequence tetrapeptide FTGN, preferablyhaving molecular weight lower than 2000 Daltons, and exhibiting saidprotective activity, is a part of this invention. Said variant maycomprise an alteration in the side chain of one amino acid in saidtetrapeptide resulting either from in vivo mutation or from chemicalmodification in vitro. Said oligomer may comprise a dimer or oligomer ofsaid tetrapeptide FTGN.

In still another embodiment, this invention is directed to a variant,modification, or a derivative of tetrapeptide FTGN, to itsphysiologically acceptable salt, ester or amide that stimulates theproliferation in vitro of bone marrow cell precursors (and/or bonemarrow stem cells). Donor bone marrow cells precursors (and/or marrowstem cells) that are treated with the oligopeptide beforetransplantation can thus proliferate after their transplantation into anirradiated mammal recipient (Example 7).

In another embodiment, this invention provides an oligopeptide extractedfrom tortoise spleen, which enhances hemopoiesis in a mammal bystimulating ex vivo/in vitro BMC-precursors (and/or bone marrow stemcells). Thus, donor BMC-precursors (and/or bone marrow stem cells) canbe ex vivo treated with the oligopeptide of the invention beforetransplantation into said mammal. An oligopeptide of this embodiment ofthe present invention protects an irradiated mammal and enhances itssurvival rate by exposing its own hemopoietic cells, or donor'shemopoietic cells to the effect of said oligopeptide.

As used herein, “precursors or progenitor cell” refers to any somaticcell, which has the capacity to generate fully differentiated,functional progeny by differentiation and proliferation. Hematopoieticprogenitor cells include those cells, which are capable of successivecycles of differentiating and proliferating to yield up to eightdifferent mature hematopoietic cells lineages. At the most primitive orundifferentiated end of the hematopoietic spectrum, hematopoieticprogenitor cells include the hematopoietic “stem cells.” These rarecells, which represent 1 in 10,000 to 1 in 100,000 of cells in the bonemarrow, each have the capacity to generate >10¹³ mature blood cells ofall lineages and are responsible for sustaining blood cell productionover the life of an animal.

A “hematopoietic stem/progenitor cell”, is a cell which is able todifferentiate to form a more committed or mature blood cell type. A“hematopoietic stem cell” or “stem cell” is one that is specificallycapable of long-term engraftment of a lethally irradiated host.

It is an object of the present invention to provide a method forenhancing the proliferation and/or differentiation and/or maintenance ofprimitive hematopoietic cells. Such a method may be useful for enhancingrepopulation of hematopoietic stem cells and thus mature blood celllineages. This is desirable where a mammal has suffered a decrease inhematopoietic or mature blood cells as a consequence of an accident,radiation or chemotherapy.

In a specifically preferred embodiment, the composition of the inventionis intended for supporting bone marrow transplantation. This effect isdue to the activity of the oligopeptide that increases proliferation ofstem cells, accelerating the hematological reconstruction upon bonemarrow transplantation and increasing the cellularity of bone marrow.

As described in Examples 4 and 5, the oligopeptide of the invention hasbeen found to enhance hematopoietic reconstruction. The aim of all BMTsis to replace the host hematopoietic stem cells injured. These stemcells can replicate repeatedly and differentiate to give rise to thewhole variety of cells.

In addition, the oligopeptide described herein may be used in thepreparation of pharmaceutical compositions enhancing proliferation oftransplanted stem cells enabling successful transplantation even whenusing a reduced donor's cell number. Increasing the number ofhematopoietic stem cells can be achieved by ex vivo/mL vitro treatmentof donor's cells prior to transplantation or by in vivo treatment ofrecipient prior or concomitant with the BM transplant procedure.

More specifically, the invention provides for the use of thistetrapeptide in the preparation of a pharmaceutical composition forsupporting bone marrow transplantation. This effect is due to theactivity of the oligopeptide in increasing proliferation of stem cells,accelerating the hematological reconstruction upon bone marrowtransplantation and increasing the cellularity of bone marrow.

In another aspect, the present invention relates to the use of the abovedescribed oligopeptide in the preparation of a pharmaceuticalcomposition for enhancement of bone marrow transplant success,hematopoietic reconstruction, bone marrow re-population particularlyafter high dose of cytotoxic exposure, said such exposure results from aconventional chemotherapy or irradiation treatment or an accidentalevent. Bone marrow transplant donor cells may be from autologous origin(autologous stem cells) or allogeneic from a compatible sibling or amatched unrelated donor.

The pharmaceutical compositions of the invention comprise as activeingredient an oligopeptide as described above, in a pharmaceuticallyacceptable carrier, excipient or stabilizer, and optionally othertherapeutic constituents. Acceptable carriers, excipients or stabilizersare non-toxic to recipients at the dosages and concentrations employed,and include buffers, such as phosphate buffered saline and likephysiologically acceptable buffers, and more generally all suitablecarriers, excipients and stabilizers known in the art, e.g., for thepurposes of adding flavors, colors, lubrication, or the like to thepharmaceutical composition.

Therapeutic formulations of the oligopeptide are prepared for storage bymixing this tetrapeptide having the desired degree of purity withoptional physiologically acceptable carriers, excipients, proteaseinhibitors or stabilizers. For ex vivo and in vitro treatment thetetrapeptide may be stored lyophilized or frozen after reconstitution insterile water or other physiological buffer.

Carriers may include starch and derivatives thereof, cellulose andderivatives thereof, e.g., microcrystalline cellulose, Xantham gum, andthe like. Lubricants may include hydrogenated castor oil and the like.

A preferred buffering agent is phosphate-buffered saline solution (PBS),which solution is also adjusted for osmolarity.

A preferred pharmaceutical formulation is one lacking a carrier. Suchformulations are preferably used for administration by injection,including intravenous injection.

The pharmaceutical composition of the invention may comprise additionalactive agents selected from growth factors, anti-rejection or toleranceinducing agents. Additional included active agents of the compositionmay be selected from the analgesic, antibiotic, anti-inflammatory,antineoplastic, cyto-protectant, glucocorticoid, hematopoietic, andimmunosuppressant groups.

The preparation of pharmaceutical compositions is well known in the artand has been described in many articles and textbooks, see e.g.,Remington's Pharmaceutical Sciences, Gennaro A. R. ed., Mack PublishingCompany, Easton, Pa., 1990, and especially pages 1521-1712 therein.

The oligopeptide or the pharmaceutical compositions to be used for invivo administration must be sterile. This is readily accomplished byfiltration through sterile filtration membranes, prior to or followinglypophilization and reconstitution. Oligopeptide may be stored insolution. Therapeutic oligopeptides compositions generally are placedinto a container having a sterile access port, for example, anintravenous solution bag or vial having a stopper pierceable by ahypodermic injection needle.

Any suitable route of administration may be employed for providing amammal, especially a human, with an effective dosage of a tetrapeptideof this invention.

The pharmaceutical compositions of the invention can be prepared indosage units forms. The dosage forms may also include sustained releasedevices. The compositions may be prepared by any of the methods wellknown in the art of pharmacy. Such dosage forms encompassphysiologically acceptable carriers that are inherently non-toxic andnon-therapeutic. Examples of such carriers include ion exchangers,alumina, aluminum stearate, lecithin, serum proteins, such as humanserum albumin, buffer substances such as phosphates, glycine, sorbicacid, potassium sorbate, partial glyceride mixtures of saturatedvegetable fatty acids, water, salts, or electrolytes such as protaminesulfate, disodium hydrogen phosphate, potassium hydrogen phosphate,sodium chloride, zinc salts, colloidal silica, magnesium trisilicate,polyvinyl pyrrolidone, cellulose-based substances, and PEG.

For all administrations, conventional depot forms are suitably used.Such forms include for example, microcapsules, nano-capsules, liposomes,inhalation forms, nose sprays and sustained-release preparations.

Suitable examples of sustained-release preparations includesemi-permeable matrices of solid hydrophobic polymers containing theoligopeptide according to the invention, which matrices are in the formof shaped articles, e.g. films, or micro-capsules. Examples ofsustained-release matrices include polyesters, hydrogels, polylactidesas described by, (U.S. Pat. No. 3,377,919), copolymers of L-glutamicacid and ethyl-L-glutamate, non-degradable ethylene-vinyl acetate,degradable lactic acid-glycolic acid copolymers such as the LupronDepots™ (injectable microspheres composed of lactic acid-glycolic acidcopolymer and leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid.While polymers such as ethylenevinyl acetate and lactic acid-glycolicacid enable release of molecules for over 100 days, certain hydrogelsrelease proteins for shorter time periods.

Sustained-release oligopeptide and particularly the FTGN compositionsmay be liposomally entrapped. Liposomes containing this oligopeptide areprepared by methods known in the art, such as described in Eppstein, etal., Proc. Natl. Acad. Sci. USA (1985) Vol. 82:3688; Hwang, et al.,Proc. Natl. Acad. Sci. USA (1980) Vol. 77:4030; U.S. Pat. Nos. 4,485,045and 4,544,545. Ordinarily, the liposomes are the small (about 200-800Angstroms) unilamellar type in which the lipid content is greater thanabout 30 mol. % cholesterol, the selected proportion being adjusted forthe optimal polypeptides therapy. Liposomes with enhanced circulationtime are disclosed in U.S. Pat. No. 5,013,556.

Although all the above mentioned techniques might be use to deliver thepharmaceutical composition to a subject in need, intravenous and oraladministration may be preferred.

The pharmaceutical composition may be administered to a subject in need,in a single or multiple occasions. The “effective treatment amount” ofthe oligopeptide or the compositions of the invention is determined bythe severity of the damaged caused to the BMC (by accident orintentionally as a manner of radiotherapy) in conjunction with thetherapeutic objectives, the route of administration and the patient'sgeneral condition (age, sex, weight and other considerations known tothe attending physician).

Accordingly, it will be necessary for the therapist to titer the dosageand modify the route of administration as required to obtain the optimaltherapeutic effect. Typically, the clinician will administer theoligopeptide in increasing dosages until the desired effect is achieved.

For therapeutic applications, the oligopeptide or the pharmaceuticalcomposition useful according to the invention are administered to amammal, preferable a human, in a physiologically acceptable dosage from,including those that may be administered to a human intravenously as abolus or by continuous infusion over a period of time. Alternativeroutes of administration include intramuscular and intraperitoneal.

The preferably single dosage comprises an amount of 1-10 mg of activeingredient/Kg body weight for in vivo treatment. The preferredconcentration of the oligopeptide in the medium that stimulates BMC isand 1-10 μg/ml and this concentration is recommended for ex vivo or invitro incubation.

In a broader aspect, the present invention provides a method thatenhances the bone marrow transplant success, the hematopoieticreconstruction, and the bone marrow re-population, after being exposedto a high dose of radiation. This method comprises administering to acell or to a subject in need thereof, an effective amount of thetetrapeptide having stimulatory activity on hematopoietic cells asdescribed above, or of a composition of the invention.

A preferred embodiment relates to a method for enhancing theproliferation of hematopoietic stem/progenitor cells. According to theinvention, this method comprises the steps of exposing these cells to aneffective amount of an oligopeptide having stimulatory activity onhematopoietic cells, or to an effective amount of a compositioncomprising the same, as described above. According to the invention suchexposure is effective in enhancing the proliferation of said cells.

The term “enhancing proliferation of a cell” encompasses the step ofincreasing the extent of growth and/or reproduction of the cell relativeto an untreated cell either in vitro/ex vivo or in vivo. An increase incell proliferation in cell culture can be detected by counting thenumber of cells before and after exposure to a molecule of interest. Theextent of proliferation can be quantified via microscopic examination ofthe degree of confluency.

The method of the invention may be used as an in vivo method oftreatment, in case that the treated cells are present in a mammal.

In vivo treatment according to the invention relates to a method forre-populating blood cells in a mammal. This method comprises the stepsof administering to said mammal a therapeutically effective amount of anoligopeptide having stimulatory activity on hematopoietic cells asdescribed above, or of a composition comprising the same. Thesehematopoietic cells may be erythroid, myeloid or lymphoid cells.

“Treatment” refers to therapeutic treatment. Those in need of treatmentare mammal subjects with a low hematopoietic cell count, a consequenceof a radiation exposure resulting from a programmed medical treatment oran accident.

“Mammal” for purposes of treatment refers to any animal classified as amammal including, human, research animals, domestic and farm animals,and zoo, sports, or pet animals, such as dogs, horses, cats, cows, etc.Preferably, the mammal is human.

For treating a subject carrying a transplant, an ex vivo method may beadopted. In this method, the cells intended for transplantation areexposed to effective amount of the oligopeptide or compositions of theinvention, prior to their transplantation.

As described in Example 7, such strategy was remarkably effective. Ashort ex vivo treatment of the transplanted cells with the tetrapeptide,improved the survival rate of the irradiated mice to 100%.

A combined ex vivo/in vivo approach may provide the basis for aforward-looking strategy for (i) obtaining small stem cell preparationsfrom donors blood or marrow and (ii) enabling healthy individuals tohave their stem cells stored for a time when the cells might be neededto treat a serious condition, thus bypassing the complexity associatedwith the use of allogeneic BMT.

This invention is also directed to a method of reducing the detrimentaleffect of ionizing radiation on tissue or body of a subject, comprisingexposing hemopoietic cells to oligopeptide derived from tortoise,wherein said exposing may be carried out ex vivo, in vitro, or in vivo.In vitro/ex vivo exposure may comprise treating donor's hemopoieticcells with said oligopeptide. In vivo exposure may comprise treatinghemopoietic cells of the irradiated subject with said oligopeptide,wherein said oligopeptide is administered to said subject.

In another embodiment of this invention, a method of reducing thedetrimental effect of ionizing radiation on tissue or body of a subjectcomprises a synthetic oligopeptide comprising sequence FTGN.

In one aspect, this invention provides a peptide comprising sequenceFTGN, preferably a peptide consisting of the sequence FTGN, particularlyfor use as a radioprotective agent which can be administered before theradiation, wherein said radiation can be a part of a therapy. In anotheraspect, this invention provides a peptide comprising or consisting ofthe sequence FTGN for use as a radioprotective agent which can beadministered after the irradiation, wherein said irradiation can be anaccidental event. A composition comprising said peptide may beadministered by injection, for example intravenously, intraperitoneally,subcutaneously, comprising a dose of about 1-10 mg/kg body weight.

The term radiation used in the invention refers to any high-energyradiation source, said such radiation comes from x-rays, gamma rays, orparticles such as neutrons and electrons. Radiation may come from amachine outside the body (external-beam radiation therapy), or it maycome from radioactive material inserted into the body near cancer cells(internal radiation therapy, implant radiation, or brachytherapy). Italso may include systemic radiation therapy which uses a radioactivesubstance, such as a radiolabeled monoclonal antibody, that circulatesthroughout the body (radiotherapy).

This invention further relates to the use of an oligopeptide extractedfrom tortoise spleen or a synthetic peptide homologous thereto, in thepreparation of a pharmaceutical composition or medicament for treatingor preventing damage to a tissue or body caused by cytotoxic agentsselected from ionizing radiations and cytotoxic chemicals. In oneembodiment of the use according to this invention, the cytotoxic agentcomprises a radiation that is a part of a therapy or accidental. Inanother embodiment, said cytotoxic agent is a part of chemotherapy.

The pharmaceutical composition of the invention may therefore beintended for increasing the white blood cells (WBC), hematopoietic stemcells in peripheral blood (PBL), and overall bone marrow cellularity.

The oligopeptide or the composition of the invention are useful in invivo or ex vivo enhancing proliferation and/or differentiation and/ormaintenance of hematopoietic stem/progenitor cells, expand population ofthese cells and enhance repopulation of such cells and blood cells ofmultiple lineages in a mammal.

It would therefore be of therapeutic importance to use small peptidessuch as the oligopeptide described in the present application, thatstimulate post-BMT hemopoietic reconstruction by enhancing in vivo, exvivo and/or in vitro the hemopoietic microenvironment.

Throughout this specification and the claims which follow, unless thecontext requires otherwise, the word “comprise”, and variations such as“comprises” and “comprising”, will be understood to imply the inclusionof a stated integer or step or group of integers or steps but not theexclusion of any other integer or step or group of integers or steps.

The invention will be further described and illustrated in the followingexamples. The following examples are representative of techniquesemployed by the inventors in carrying out aspects of the presentinvention. It should be appreciated that while these techniques areexemplary of preferred embodiments for the practice of the invention,those of skill in the art, in light of the present disclosure, willrecognize that numerous modifications can be made without departing fromthe intended scope of the invention.

EXAMPLES

Materials and Methods

1. Tortoise Spleens Extract Preparation

Spleen extracts were prepared from 6-10 years old Testudo horsfieldi,Tortoise from Uzbekistan and Kazakhstan.

Crude extract of tortoise spleen was prepared from 200 tortoises. Theanimals were sacrificed by decapitation, and the spleens were collectedin a dish placed into liquid nitrogen bath. Deep frozen tissue wascrushed to powder. One liter of PBS containing protease inhibitorcocktail was added to powder, placed at 4° C. for 24 hours, andcentrifuged twice at 4400 g for 30 min. The supernatant was lyophilized.To remove hemoglobin from the above crude extract, the powder extractwas dissolved in distilled water to a concentration of 50 mg/ml andcooled in ice/water bath. Ethanol/chloroform (2/1) was added drop-wiseto the mixture, 2 ml per 1 ml of mixture. After mixing for 20 min, themixture was centrifuged at 0° C. for 30 min. Supernatant was separatedand lyophilized. The powder was dissolved in superpure distilled waterto a concentration of 40 mg/ml. After filtration by Syringe filter(Nylon 0.45 mcm) the mixture was applied onto a preparative HPLC C18column (Vydac 218TP, 250×12 mm) (FIG. 1).

2. Tortoise Spleens Extract Fractions Purification

The peaks from 20 batches of HPLC described above (FIG. 1) were pooledand characterized by the MALDI-TOFF mass spectra analysis and by NMR,and by measuring their biological activities.

Active fractions were re-purified on preparative HPLC column C18 toyield six peaks (denoted a1 to a6 in FIG. 4). Proteinaceous fraction a5was found to be most active. The results of TLC (Silica Gel 60F254) offractions a4 to a6 were developed by ninhidrin, together with free aminoacids Glu, Val, Gly, and Met, are shown in FIG. 5. Fraction a5 indicatesthe presence of a heavier peptidic component (near the start). Thisfraction was collected from several runs, and pooled. A peptide ofmolecular weight 437.45 (MS) was identified. Sequencing by the Edmannmethod (carried out at the Weizmann Institute in Rehovot, Israel) showedtetrapeptide FTGN.

3. Tetrapeptide FTGN Synthesis

Tetrapeptide FTGN was synthesized by the solid phase peptide synthesismethod, using the 9-fluorenylmethoxycarbonyl (FMOC) strategy. FITC wasassembled with a handle (e-aminocaproic acid). The peptides were thencleaved from the resin with trifluoroacetic acid solution. Finally thepeptides were purified on reverse phase HPLC, dry freeze and stored at−10° C.

The oligopeptide was synthesized at Sigma-Aldrich (Israel).

4. Animals

Five weeks old male C57Bl/6J mice were used as bone donors for BMCpreparation. Mice from the same batches were irradiated and used as BMCrecipients.

Mice were subjected to sub-lethal and lethal radiation doses (from4.5-10 Gy) according to the experiment design.

5. Biological Methods

The biological activities of this synthesized oligopeptide were studiedat the Department of Hematology of the Hadassah University Hospital,Jerusalem.

a) Bone Marrow Cell Collection

Mice femurs were removed and placed in PBS solution. Cell suspensions ofbone marrow were prepared by washing each cavity of the femur with 2.0ml PBS with a sterile syringe and 26-gauge needle. Bone marrow cellcounts were obtained using a hemocytometer. Viability was assessed byTrypan blue.

b) Bone Marrow Cells Transplant

Bone marrow cells (BMC) were transplanted into mice by intravenousinjection, usually 60,000 cells in 0.3 ml of PBS buffer after 2 hoursincubation at 37° C. with the examined agent.

c) Mice Spleen Colonies Assessment

Mice spleen colonies (CFUs) were counted using the method of Till andMcCulloch [Till J. E. and McCulloch E. A. Radiation Res. (1961) 14: 213]on 9th, 11th and 14th days after irradiation.

CFUc were assayed in semi-solid medium using 2×10⁴ cells/plate.

Example 1

Biological Activity of the HPLC Fractions Isolated from Spleen Extract

The tortoise spleen extract HPLC purified fractions (FIG. 1) were testedfor their biological activity. Irradiated mice (6 Gy) were injectedintraperitoneally with 2 μg of the different fractions. Thehematopoietic activity in these mice was examined by the number ofspleen endocolonies formation and by bone marrow smear preparations atthe 14th day post-irradiation. Fraction d stimulated the growth ofspleen endocolonies but showed no influence on the BMC. Injection offractions e or f resulted in the same lack of effect as seen in thecontrol mice group injected with PBS; no biological influence wasobserved. Fraction g did not increase the number of the spleen coloniesbut showed some influence on the BMC. Fraction h exerted activity onboth tested parameters: it helped in the repopulation of BM andincreased the number of spleen forming colonies (FIG. 2). Further HPLCmass spectra analysis of the fractions g and h revealed a peak similarto a 8450 Daltons polypeptide. Sequencing analysis of this polypeptiderevealed that it resembled the ubiquitin molecule, well known to beinvolved in many cell processes such as in the regulation of the cellcycle, DNA repair, embryogenesis, regulation of transcription, andapoptosis.

Ex vivo treatment of donor BM cells with purified spleen extractfraction a and transplantation into irradiated mice, showed asignificant influence on the BM cell restoration as seen in FIG. 3.Additional purification of fraction a (described above in methods, FIG.4) identified the subfraction a5, as the responsible for the biologicalactivity previously described. Furthermore, irradiated mice transplantedwith BMC treated with purified subfraction a5, increased their survivalrate as evaluated 30 days post-irradiation (FIG. 19). TLC (FIG. 5) andsequencing analysis of the a5 subfraction identified the tetrapeptideFTGN as the active factor.

Example 2

Physical Characteristic of FTGN Oligopeptide

Dry freeze long term storage of the FTGN oligopeptide preserved itsstructural (FIG. 6 a) and biological activities. The dry frozentetrapeptide conserved its physical characteristics after reconstitutionin purified water or serum (FIG. 6 c) and was stable after four hours ofincubation at 37° C.

Example 3

Biological Activity of FTGN Tetrapeptide Tested In Vitro

Bone marrow cells from normal (non irradiated) mice (4-6×10⁶ cells/ml)were incubated for 2 hours with 10 μg/ml tetrapeptide; the cells werewashed and cloned in cytokine-supplemented methylcellulose semi-solidmedium.

Myeloid colonies formed after two weeks were counted. As seen in FIG. 7a, a short in vitro incubation of bone marrow cells with thetetrapeptide resulted in an increased colony number.

Similarly, in vivo and ex vivo irradiated cells (4.5 Gy intensity)incubated for 2 hours with 10 μg/ml FTGN tetrapeptide and treated asdescribed above, formed a larger number of colonies compared to cellsthat underwent the same irradiation treatment but were not brought incontact with the tetrapeptide (SEQ ID: No. 1) (FIGS. 7 b and 7 c).

The presence of the tetrapeptide stimulates hematopoiesis and helpsrestore the growth of damaged cells lineages.

Example 4

Ex Vivo BMC Treatment with FTGN Tetrapeptide

In an attempt to assess the most favorable ex vivo treatment conditionsneeded in order to obtain a maximal bone marrow recovery of BMtransplanted irradiated recipients, the following experiments wereperformed.

Donor normal BMC (4-6×10⁶ cells/ml) from C57 Bl/6J mice were incubatedwith different concentrations of FTGN tetrapeptide (0.05; 0.1; 0.5; 1.0;10 and 50 μg/ml) for two hours. Whole body irradiated (7.0 Gy) C57Bl/6Jmice were intravenously injected with 6×10⁴ treated or control cells.The recovery of the hematopoietic system in the transplanted mice wasevaluated from the 4^(th) to the 30^(th) day post-irradiation.

Spleen Morphology, Weight and CFUs:

No difference in the spleen weight was observed in the 4^(th) day aftertransplantation between the control or the experimental groups (FIGS. 8and 9).

By the 9^(th) day the spleen weight, as well as the spleen coloniesnumber, was augmented in mice transplanted with FTGN tetrapeptidetreated cells (FIGS. 8 a and 10) in comparison to the spleens of micetransplanted with non-treated cells (FIGS. 8 b and 10).

In contrast, by the 16^(th) day, the spleen weight of the experimentalgroup was lower and the spleen surface was smoother relatively to thecontrol group (FIG. 8 b).

These results suggest that FTGN tetrapeptide treatment may wellaccelerate hematopoietic recovery of the spleen. Mice transplanted withFTGN-treated cells demonstrated an early increase in spleen size andfaster development of spleen colonies followed by an earliernormalization of the spleen size.

The total number of nucleated bone marrow cells per femur on the 16^(th)day was doubled in the animals transplanted with BM cells treated withFTGN oligopeptide (FIG. 11); colony-forming cells (CFUbm) in the bonemarrow assayed in semi-solid medium on the 9th and the 16th day showedan improvement when low concentrations of the oligopeptide were used(FIG. 12). The peripheral blood white cells counts (WBC) calculated onthe 16^(th) day were improved in all the mice transplanted with FTGNtreated cells (FIG. 13).

Example 5

Optimal FTGN Tetrapeptide Treatment Concentration

In order to elucidate the optimal FTGN tetrapeptide treatmentconcentration needed to achieve maximum recovery of radiation damagedBMC, two sets of donor cells were examined. Donor BMC (4-6×10⁶ cells/ml)from normal untreated C57 Bl/6J mice and from in vivo irradiated mice(4.5 Gy) collected 24 hours post-irradiation, were incubated for twohours with different concentrations of FTGN tetrapeptide (0.05; 0.1;0.5; 1.0; 10 and 50 μg/ml). Whole body irradiated (7.0 Gy) C57Bl/6J micewere intravenously injected with 6×10⁴ of each different donor treatedcells or with control cells. The recovery of the hematopoietic system inthe transplanted mice was evaluated from the 4th to the 30^(th) daypost-irradiation.

The highest spleen colony forming units (CFUs) count, by the 9^(th) day(FIG. 14), and the greatest number of nucleated cell in BM oftransplanted mice (4, 9 and 16 days after irradiation; FIG. 15) wasobtained with a dose of 1-10 μg/ml of FTGN tetrapeptide ex vivotreatment.

The FTGN tetrapeptide ex vivo treatment accelerated the BM repopulationactivity after transplant regardless if the donor's cells were normal ororiginated in an irradiated mouse (FIG. 16).

The ex vivo treatment of donor cells with the tetrapeptide increasedtheir potential to repopulate the BM and spleen of lethally irradiatedmice.

Example 6

In Vivo Treatment with FTGN Tetrapeptide

Irradiated mice (6.5 Gy) were treated with one or two intravenous 50 μgFTGN tetrapeptide injections (2 or 2 and 24 hours post-irradiation). BMnucleated cells count (FIG. 17) and CFUc (FIG. 18) were examined onemonth later. Treatment with the FTGN tetrapeptide increased the BMrepopulation in mice damaged by ionizing radiation.

Example 7

Survival of Lethal Irradiated Mice Transplanted with BM Cells Treatedwith Spleen Extract Fraction a5 or FTGN Tetrapeptide

Irradiated mice (8 Gy) were transplanted with donor BM cells treatedwith the purified spleen extract subfraction a5. Mice survival, asresumed after 30 days, was improved by about 30% in the mice groupinjected with the treated BMC (FIG. 19).

In a further experiment, using more extreme irradiation conditions andthe purified FTGN oligopeptide, even more remarkable results wereachieved.

Mice irradiated with a high lethal dose (10 Gy) were injected with 6×10⁴cells previously incubated for one hour with 10 μg/ml FTGN tetrapeptide.Mice survival was surveyed for a 30 days period post-transplant. As seenin FIG. 20, the FTGN oligopeptide treatment ensured a 100% survival.

The results show a remarkable hemopoietic activity of the peptideaccording to this invention.

While this invention has been described in terms of some specificexamples, many modifications and variations are possible. It istherefore understood that within the scope of the appended claims, theinvention may be realized otherwise than as specifically described.

1-44. (canceled)
 45. An oligopeptide comprising the amino acid sequenceFTGN (SEQ ID: No. 1).
 46. An isolated oligopeptide, consisting of theamino acid sequence FTGN (SEQ ID: No. 1) and pharmaceutically acceptablesalts thereof.
 47. A synthetic oligopeptide consisting of the amino acidsequence FTGN (SEQ ID: No. 1) and pharmaceutically acceptable saltsthereof.
 48. An oligopeptide according to claim 45, which stimulates theproliferation of mammalian hemopoietic cells, or its variants,modifications and derivatives wherein said variants, modifications andderivatives maintain the ability of said oligopeptide to stimulate theproliferation of mammalian hemopoietic cells, and physiologicallyacceptable salts, esters and amides of said oligopeptide and functionalmodifications or derivatives thereof.
 49. An oligopeptide according toclaim 48, wherein said variant comprises an alteration in the side chainof an amino acid of said oligopeptide resulting from either in vivomutation or from chemical modification in vitro.
 50. An oligopeptideaccording to claim 48, wherein said variant comprises a dimer ormultimer of said oligopeptide.
 51. An oligopeptide according to claim48, wherein said modification or derivative comprises a chemicalmodification of side chains of the amino acids, or modification of theterminal carboxyl or amino groups.
 52. An oligopeptide according toclaim 48, wherein said modification comprises alkylation, acylation,amidation, or esterification.
 53. An oligopeptide according to claim 48,wherein said modification modulates the biological activity of saidoligopeptide, and/or improves its stability.
 54. An oligopeptideaccording to claim 48, wherein said modification improves the in vivostability of said oligopeptide.
 55. A protective composition comprisinga an oligopeptide of claim 45 or physiologically acceptable salts,esters, and amides of said oligopeptide or its functional variants,modifications, and derivatives, for reducing the detrimental effect of acytotoxic factor selected from ionizing radiation and cytotoxicchemicals on a tissue or subject exposed thereto.
 56. A protectivecomposition according to claim 55, wherein the exposure of tissue orbody to ionizing radiation is accidental or a part of radiation therapy.57. A protective composition according to claim 55, wherein the exposureof tissue or body to said cytotoxic chemical is accidental or a part ofchemotherapy.
 58. A protective composition according to claim 55, for invivo treatment, preferably by injection.
 59. A protective compositionaccording to claim 55, for in vitro or ex vivo treatment of hemopoieticcells.
 60. A pharmaceutical composition comprising an oligopeptide asdefined in claim 45 or physiologically acceptable salts, esters, andamides of said oligopeptide or its functional variants, modifications,and derivatives, and further comprising a pharmaceutically acceptablecarrier, excipient and/or diluent.
 61. A pharmaceutical compositioncomprising as active ingredient a tetrapeptide consisting of the aminoacid sequence FTGN (SEQ ID: No. 1), a functional derivative thereof or apharmaceutically acceptable salt, ester or amide thereof.
 62. Apharmaceutical composition according to claim 60, further comprising anadditional active agent selected from growth factor and anti-rejectionagent or tolerance inducing agent.
 63. A pharmaceutical compositionaccording to claim 60, further comprising an additional active agentselected from the group consisting of analgesics, antibiotics,anti-inflammatories, antineoplastics, cyto-protectants,gluco-corticoids, hematopoietics, and immunosuppressants.
 64. A methodfor reducing the detrimental effect of a cytotoxic factor selected fromionizing radiation and cytotoxic chemicals on a mammalian subjectexposed thereto, comprising administering to said subject anoligopeptide of claim 45 or a physiologically acceptable salt, ester, oramide thereof or a functional variant, modification, or derivativethereof.
 65. A method for reducing the detrimental effect of a cytotoxicfactor selected from ionizing radiation and cytotoxic chemicals on atissue or body of a mammal exposed thereto, comprising the steps of:obtaining hemopoietic cells of said mammal; ex vivo treatment of saidhemopoietic cells with an oligopeptide as defined in claim 45 or withphysiologically acceptable salts, esters, and amides thereof or withfunctional variants, modifications, and derivatives thereof; andre-implanting said cells into said mammal.
 66. A method for reducing thedetrimental effect of a cytotoxic factor selected from ionizingradiation and cytotoxic chemicals on a tissue or body of a mammalexposed to said factor, comprising the steps of: treating donor'shemopoietic cells with an oligopeptide of claim 45 or withphysiologically acceptable salts, esters, and amides thereof or withfunctional variants, modifications, and derivatives thereof; andtransplanting said donor's cells into said mammal.
 67. A method forreducing the detrimental effect of a cytotoxic factor selected fromionizing radiation and cytotoxic chemicals on a tissue or body of amammal exposed to said factor, comprising administering to said mammalan oligopeptide as defined in claim 64 or physiologically acceptablesalts, esters, and amides thereof or functional variants, modifications,and derivatives thereof.
 68. A method according to claim 64, wherein theexposure of said tissue or body to ionizing radiation is accidental or apart of a radiation therapy.
 69. A method according to claim 67, whereinthe exposure of said tissue or body to a cytotoxic chemical isaccidental or due to chemotherapy.
 70. A method according to claim 67,wherein said cells are treated with said composition of matter or saidpeptide or said derivative after the exposure to said cytotoxic factor.71. A method according to claim 64, wherein said cells are treated withsaid composition of matter or said peptide or said derivative before theexposure to said cytotoxic factor.
 72. An oligopeptide of claim 45 orphysiologically acceptable salts, esters, and amides thereof orfunctional variants, modifications, and derivatives thereof, comprisingthe step of using the oligopeptide as a medicament.
 73. An oligopeptideof claim 45 or physiologically acceptable salts, esters, and amidesthereof or functional variants, modifications, and derivatives thereof,comprising the step of using the oligopeptide as a medicament forreducing the detrimental effect of a cytotoxic factor selected fromionizing radiation and cytotoxic chemicals on a tissue or body of amammal exposed thereto.
 74. A method comprising providing a tetrapeptidehaving the amino acid sequence FTGN (SEQ ID: No. 1), or physiologicallyacceptable functional derivatives thereof comprising alkylation,acylation, amidation, or esterification, or pharmaceutically acceptablesalts of said tetrapeptide or its derivatives, and using the step ofusing the tetrapeptide as a medicament.
 75. A method comprisingproviding an oligopeptide comprising the amino acid sequence FTGN (SEQID: No. 1), and using the step of using the oligopeptide as a protectiveagent.